More than 70% of all agricultural pests are insects in the order Lepidoptera, which, unlike other related insect orders, are not very sensitive to RNAi, limiting genetic studies of this insect group. However, the reason for this distinct lepidopteran characteristic is unknown. Previously, using transcriptome analysis of the Asian corn borer , we identified a gene, termed, that is up-regulated in response to dsRNA. Here we report that this Lepidoptera-specific gene encodes a nuclease that contributes to RNAi insensitivity in this insect order. Its identity was experimentally validated, and sequence analysis indicated that encodes a previously uncharacterized protein with homologous sequences in seven other lepidopteran species. Its computationally predicted three-dimensional structure revealed a high structural similarity to human exonuclease I. Exposure to dsRNA in strongly up-regulated this gene's expression, and the protein could digest single-stranded RNA (ssRNA), dsRNA, and dsDNA both and Of note, we found that this up-regulation of expression is faster than that of the gene encoding the key RNAi-associated nuclease knockdown in significantly enhanced RNAi efficiency. Moreover, overexpression in suppressed RNAi efficiency. Finally, knockdown significantly increased the amount and diversity of small RNAs. Therefore, we renamed this protein RNAi efficiency-related nuclease (REase). In conclusion, we propose that REase may explain why lepidopterans are refractory to RNAi and that it represents a target for further research of RNAi efficiency in this insect order.

Double-stranded RNA (dsRNA)-induced genes are usually related to RNA interference (RNAi) mechanisms and are involved in immune-related pathways. In a previous study, we found a lepidopteran-specific nuclease gene REase that was up-regulated by dsRNA and that affected RNAi efficiency in Asian corn borer (Ostrinia furnacalis). In this study, to verify the function of REase, the homologous gene HaREase in cotton bollworm (Helicoverpa armigera) was knocked out using CRISPR/Cas9 system. We found that the midgut epithelium structure was apparently not affected in the ΔHaREase mutant [Knock out (KO)]. Transcript sequencing results showed that most of the known insect immune-related genes were up-regulated in KO. When second instar larvae were fed artificial diet with Cry1Ac, a protoxin from Bacillus thuringiensis (Bt), in sublethal doses (2.5 or 4 μg/g), the growth rate of KO was repressed significantly. The dsRNA stability was also enhanced in midgut extraction of KO; however, RNAi efficiency was not obviously improved compared with the wild type (WT). The KO and WT were injected with dsEGFP (Enhanced green fluorescent protein) and subjected to transcriptome sequencing. The results showed that the expression levels of 14 nuclease genes were enhanced in KO after the dsRNA treatment. These findings revealed that HaREase expression level was not only related with dsRNA stability, but also with Bt resistance in cotton bollworm. When HaREase was knocked out, other immune- or nuclease-related genes were enhanced significantly. These results remind us that insect immune system is complex and pest control for cotton bollworm is an arduous task.